RESUMEN
The L. fermentum U-21 strain, known for secreting chaperones into the extracellular milieu, emerges as a promising candidate for the development of novel therapeutics termed disaggregases for Parkinson's disease. Our study focuses on characterizing the secreted protein encoded by the C0965_000195 locus in the genome of this strain. Through sequence analysis and structural predictions, the protein encoded by C0965_000195 is identified as ClpL, homologs of which are known for their chaperone functions. The chaperone activity of ClpL from L. fermentum U-21 is investigated in vivo by assessing the refolding of luciferases with varying thermostabilities from Aliivibrio fischeri and Photorhabdus luminescens within Escherichia coli cells. The results indicate that the clpL gene from L. fermentum U-21 can compensate for the absence of the clpB gene, enhancing the refolding capacity of thermodenatured proteins in clpB-deficient cells. In vitro experiments demonstrate that both spent culture medium containing proteins secreted by L. fermentum U-21 cells, including ClpL, and purified heterologically expressed ClpL partially prevent the thermodenaturation of luciferases. The findings suggest that the ClpL protein from L. fermentum U-21, exhibiting disaggregase properties against aggregating proteins, may represent a key component contributing to the pharmabiotic attributes of this strain.
RESUMEN
Several clades of luminescent bacteria are known currently. They all contain similar lux operons, which include the genes luxA and luxB encoding a heterodimeric luciferase. The aldehyde oxygenation reaction is presumed to be catalyzed primarily by the subunit LuxA, whereas LuxB is required for efficiency and stability of the complex. Recently, genomic analysis identified a subset of bacterial species with rearranged lux operons lacking luxB. Here, we show that the product of the luxA gene from the reduced luxACDE operon of Enhygromyxa salina is luminescent upon addition of aldehydes both in vivo in Escherichia coli and in vitro. Overall, EsLuxA is much less bright compared with luciferases from Aliivibrio fischeri (AfLuxAB) and Photorhabdus luminescens (PlLuxAB), and most active with medium-chain C4-C9 aldehydes. Crystal structure of EsLuxA determined at the resolution of 2.71 Å reveals a (ß/α)8 TIM-barrel fold, characteristic for other bacterial luciferases, and the protein preferentially forms a dimer in solution. The mobile loop residues 264-293, which form a ß-hairpin or a coil in Vibrio harveyi LuxA, form α-helices in EsLuxA. Phylogenetic analysis shows EsLuxA and related proteins may be bacterial protoluciferases that arose prior to duplication of the luxA gene and its speciation to luxA and luxB in the previously described luminescent bacteria. Our work paves the way for the development of new bacterial luciferases that have an advantage of being encoded by a single gene.
RESUMEN
There are many facts about the possible role of gamma-aminobutyric acid (GABA) in the development and differentiation of cells not only in nervous but also in muscle tissue. In the present study a primary culture of rat skeletal muscle myocytes was used to evaluate the correlation between the content of GABA in the cytoplasm and the processes of myocyte division and their fusion into myotubes.The effect of exogenous GABA on the processes of culture development was also estimated. Since the classical protocol for working with myocyte cultures involves the use of fetal bovine serum (FBS) to stimulate cell division (growth medium) and horse serum (HS) to activate the differentiation process (differentiation medium), the studies were carried out both in the medium with FBS and with HS. It was found that cells grown in medium supplemented withFBS contain more GABA compared to cultures growing in medium supplemented with HS. Addition of exogeneous GABA leads to a decrease in the number of myotubes formed in both media, while the addition of an amino acid to the medium supplemented with HS had a more pronounced inhibitory effect. Thus, we have obtained data indicating that GABA is able to participate in the early stages of skeletal muscle myogenesis by modulating the fusion process.
RESUMEN
Bacterial expression systems play an indispensable role in the biosynthesis of recombinant proteins. Different proteins and the tasks associated with them may require different systems. The purpose of this work is to make an expression vector that allows switching on and off the expression of the target gene during cell incubation. Several expression vectors for use in Escherichia coli cells were developed using elements of the luxR/luxI type quorum sensing system of psychrophilic bacterium Aliivibrio logei. These vectors contain A. logei luxR2 and (optionally) luxI genes and LuxR2-regulated promoter, under the control of which a target gene is intended to be inserted. The synthesis of the target protein depends directly on the temperature: gene expression starts when the temperature drops to 22 °C and stops when it rises to 37 °C, which makes it possible to fix the desired amount of the target protein in the cell. At the same time, the expression of the target gene at a low temperature depends on the concentration of the autoinducer (L-homoserine N-(3-oxohexanoyl)-lactone, AI) in the culture medium in a wide range from 1 nM to 10 µM, which makes it possible to smoothly regulate the rate of target protein synthesis. Presence of luxI in the vector provides the possibility of autoinduction. Constructed expression vectors were tested with gfp, ardA, and ardB genes. At maximum, we obtained the target protein in an amount of up to 33% of the total cellular protein. KEY POINTS: ⢠A. logei quorum sensing system elements were applied in new expression vectors ⢠Expression of target gene is inducible at 22 °C and it is switched off at 37 °C ⢠Target gene expression at 22 °C is tunable by use different AI concentrations.